Although previous research suggested that u-PA was involved in some tumor metastasis such as lung-specific metastasis, the role of u-PA for peritoneal metastasis in gastric cancer is still unclear. The aim of this study was to explore whether selective pharmacological blockade of u-PA is able to affect the peritoneal metastasis of gastric cancer both In the present study, we evaluated the effects and explored the anti-tumor mechanisms of amiloride, a selective u-PA inhibitor, on a panel of gastric cancer cell lines and in a murine model of human gastric cancer MKN45.vitro, compared with controls, amiloride could not only significantly down-regulate the mRNA expression and protein level of u-PA from MKN45 cells with dose dependence but also inhibit the adhesion of HMrSV5 cells, migration and invasion of MKN45 cells.The study showed that amiloride significantly inhibited the tumor growth and prolonged the survival of the tumor-bearing mice. In The findings in our current report provide evidence that selective u-PA inhibitor amiloride has potent effects against peritoneal metastasis in gastric cancer, suggesting its possible therapeutic value for the treatment of gastric cancer. Metastasis and recurrence of peritoneal cancer, especially peritoneal metastasis in gastric cancer, which is often associated with lymphatic infiltration, is a prevalent cause of death in patients with gastric cancer in clinical practice -4. TheraMetastasis is a complex process that mediates detachment of cancer cells from a primary site, invasion into surrounding tissues, spread through the circulation, extravasion and proliferation in distant organs . The uroMoreover, in recent years, amiloride, a selective u-PA inhibitor, has been proved to have interventional effects on gastric cancer. Antisense inhibition of u-PA could reduce the spread of human ovarian cancer in mice . In thisHattiesburg, USA) and was dissolved in saline.Four to five week-old male BALB/c nude mice under SPF conditions were obtained from the Experimental Animal Center  and given free access to water and food. All experiments conformed to the animal care and use guidelines of the Institute’s Animal Care and Use Committee. Amiloride was obtained from the Sigma Company  supplemented with heat-inactivated fetal bovine serum (FBS) , 100 units/ml penicillin, and 100 units/ml streptomycin in a humid chamber at 37°C under 5% CO6 cells per dish. The incubation times after cell seeding with amiloride were 6 h, 12 h and 24 h. After incubation over these time periods, cells were treated with amiloride at concentrations of 0.01 mM, 0.1 mM or 1 mM, and RPMI 1640 with 10% FBS was used as a control. Total RNA was isolated from MKN45 cells using TRIzol reagent  according to the manufacturer’s instructions. The concentration of RNA was determined and cDNA was generated using total RNA with the Reverse Transcriptase kit . RT-PCR products were visualized through 2% agarose gels containing ethidium bromide by electrophoresis. For amplification of the desired cDNA, the specific primers were used as follows: uPA: F:5′-AGA ATT CAC CAC CAT CGA GA-3′, R:5′-ATC AGC TTC ACA ACA GTC AT-3′; GAPDH: F:5′-GAA GGTGAAGGTCGGAGT C-3′, R:5′-GAA GAT GGT GAT GGG ATT TC-3′.The MKN45 cells were added in the dishes at the amount of 2 × 10Total protein of cell lysates from MKN45 treated by amiloride were tested for the presence of u-PA. Concentration of u-PA was determined with commercial ELISA kits  according to the manufacturer’s instructions. The reported value is the mean ± SD of duplicate samples.In brief, the MKN45 cells treated by amiloride were collected, washed three times with ice-cold phosphate-buffered saline (PBS) and lysed in ice-cold lysis buffer , 0.5 mM EDTA, 1% nonidet P40 (NP40), 100 μg/ml PMSF, 1 μg/ml aprotinin, 2 μg/ml leupeptin 100 μM, sodium vanadate, PH 8.0) for 15 minutes at 1500 g centrifugation. Cellular proteins were extracted from the MKN45 treated by amiloride. u-PA activity was assessed with the u-PA Activity Assay Kit  according to the manufacturer’s instructions. Results are presented as the mean ± SD of triplicate wells.4 cells per well in 96-well multiwall plates 12 to 24 h prior to the assays. Then MKN45 cells were added in the 96-well multiwall plates at the concentration of 1 × 104 cells/ml. The adhesion assay was determined by the MTT assay [The adhesion assays we tested in this study followed a previous description . HMrSV5 TT assay . The inc%.% Inhibition rate = (A570 of control cell - A570 of treated cells)/A570 of control cells × 100Boyden chamber migration and invasion assaysin vitro migrating and invasive capacity of tumor cells was essentially the same as described in previous reports [4 in 500 μl of serum-free medium and incubated for 12 to 24 h. For the migration assay, 2×104 MKN45 cells per 250 μl were seeded on the 20% fibronectin-coated filters and incubated for 24 h. The lower chamber was filled with 300 μl of full medium. After incubation for 48 h, the cells were counted by the MTT assay [4 MKN45 cells at the concentration of 8×104 cells/ml were seeded on the 10% ice-cold Matrigel (BD Bioscience)-coated filters and incubated for 24 h. The lower chamber was filled with 300 μl of full medium. After incubation for 72 h, the number of penetrated single tumor cells and tumor cell colonies  was counted by the MTT assay [The assay procedure to measure the  reports -14. AssaTT assay . After iTT assay . Incubat%.Migration rate = A570 of lower/A570 of upper + A570 of lower × 1006 MKN45 cells were intraperitoneally injected into 4- to 5-week-old male BALB/c nude mice. The mice were randomly divided into two groups of 12 mice. Fourteen days after MKN45 implantation, the treatment groups received their first dose of amiloride dissolved in a saline solution. Amiloride dosage and administration schedules were based on our preliminary toxicologic and pharmacokinetic studies. Briefly, amiloride was given via oral administration to tumor-bearing mice at 50 mg/kg every day at the first three days of one week for a total time of four weeks. In parallel, the control group received the saline solution. General clinical observations of the mice, including determination of body weight and tumor growth (data not shown), were made twice weekly. The mice were sacrificed when they became moribund, and the sacrifice date was recorded to calculate the survival time.The tumor model used in this study has been described previously . Brieflyt-test or one way analysis of variance (ANOVA) followed by post hoc tests when appropriate. Differences in non-parametric data were evaluated by the Mann–Whitney U-test. Survival curves were statistically analyzed using Kaplan-Meier test. Data were expressed as means ± SD. A significant difference was defined as P < 0.05.Statistical analysis was performed with the SPSS software system . Parametric data were statistically analyzed by the Student’s u-PA, which by its signaling is essential for the onset of gastric cancer, plays an important role in both the human and animal model of gastric cancer -18. As sIn vitro, compared with controls, amiloride significantly decreased the adhesion of mesothelial HMrSV5 cells. This inhibition was time- and dose-dependent (Figure t Figure A.We investigated the effects of amiloride on the Boyden chamber migration of MKN45 gastric cancer cells. As shown Figure , amiloriWe analyzed the effects of amiloride on the mRNA expression of uPA in the gastric cancer cell line MKN45 Figure . As showHaving shown that amiloride suppressed tumor cell growth, we investigated its antitumor effects in a murine model of gastric cancer. As shown in Figure Treatment of human peritoneal metastasis in gastric cancer remains a major clinical problem . Metasta+ transport, competitively inhibits the catalytic activity of u-PA, without decreasing those of t-PA, or plasmin [et al. have observed that u-PA knockdown could inhibit the proliferation of cancer cells in peritoneal metastasis [et al. reported that treatment of amiloride could not only reduce the size of prostate cancer xenografts in severe combined immunodeficient mice, but also help survival [et al. also agreed that amiloride played a specific role in inhibiting u-PA activity [in vitro. In accordance to previous reports, the results showed that amiloride not only decreased both mRNA and protein production of u-PA in MKN45 gastric cancer cells, but also reduced the u-PA activity of MKN45 cell line. Amiloride inhibited MKN45-derived tumor growth and prolonged the survival of the tumor-bearing mice. Consequently, it might be concluded that the inhibition of u-PA by amiloride could suppress peritoneal metastasis in gastric cancer. Furthermore, activation of Na+/H+ exchange activity is found as a ubiquitous response to early growth factors, such as u-PA [It is reported that amiloride, an inhibitor of Na plasmin . Thus, a plasmin . u-PA is plasmin  . Lokmantastasis . Jankun survival . To furtsurvival . Ogura eactivity . We anal as u-PA . uPA/uPA as u-PA . Amilori as u-PA -33. Takein vitro, Tuck et al. had found that the same as anti-u-PA antibody and anti-u-PAR antibody, amiloride significantly inhibited migration and invasion of breast cancer cells [et al. observed that oral amiloride inhibited lung metastasis in pulmonary metastasis in the rat mammary cancer model and this effect was positively correlated with time- and dose-dependence [et al. found that oral amiloride could potently inhibit tumor growth and prolong the survival of tumor bearing mice [et al. reported that amiloride could dose- and time-dependently inhibit cancer cell metastasis [In a recent study on breast cancer cells er cells . Evans ependence . In SCIDing mice . Evans etastasis . In the in vitro. However, there was no significant effect of amiloride on migration of gastric cancer cell MKN45 peritoneal metastasis. It is interesting to further elucidate the precise mechanism of cancer cell migration and invasion in gastric cancer peritoneal metastasis mediated by u-PA inhibition.In our study, compared with controls, the selective u-PA inhibitor amiloride significantly decreased the adhesion of mesothelial HMrSV5 cells, and inhibited the adhesion of the gastric cancer cell MKN45 The authors declare that they have no competing interests.YD and HZ participated in the design of this study, and they both performed the statistical analysis. ZZ carried out the study, together with QC and MZ collected important background information, and drafted the manuscript. MZ, XW and ZZ conceived this study, participated in the design and helped to draft the manuscript. All authors read and approved the final manuscript.


The prevalence of adolescents’ obesity and overweight has dramatically elevated in China. Obese children were likely to insulin resistance and dyslipidemia, which are risk factors of cardiovascular diseases. However there was no cut-off point of anthropometric values to predict the risk factors in Chinese adolescents. The present study was to investigate glycolipid metabolism status of adolescents in Shanghai and to explore the correlations between body mass index standard deviation score (BMI-SDS) and metabolic indices, determine the best cut-off value of BMI-SDS to predict dyslipidemia.Fifteen schools in Shanghai’s two districts were chosen by cluster sampling and primary screening was done in children aged 9-15 years old. After screening of bodyweight and height, overweight and obese adolescents and age-matched children with normal body weight were randomly recruited in the study. Anthropometric measurements, biochemical measurements of glycolipid profiles were done. SPSS19.0 was used to analyze the data. Receiver operating characteristic (ROC) curves were made and the best cut-off values of BMI-SDS to predict dyslipidemia were determined while the Youden indices were maximum.Five hundred and thirty-eight adolescents were enrolled in this research, among which 283 have normal bodyweight, 115 were overweight and 140 were obese. No significant differences of the ages among 3 groups were found. There were significant differences of WC-SDS (p<0.001), triacylglycerol (p<0.05), high and low density lipoprotein cholesterol (p<0.01), fasting insulin (p<0.01) and C-peptide (p<0.001) among 3 groups. Significant difference of fasting glucose was only found between normal weight and overweight group. Significant difference of total cholesterol was found between obese and normal weight group. There was no significant difference of glycated hemoglobin among 3 groups. The same tendency was found in boys but not in girls. Only HDL-C reduced and TG increased while BMI elevated in girls. The best cut-off value of BMI-SDS was 1.22 to predict dyslipidemia in boys. The BMI cut-off was 21.67 in boys.Overweight and obese youths had reduced insulin sensitivity and high prevalence of dyslipidemia.When BMI-SDS elevated up to 1.22 and BMI was higher than 21.67 in boys, dyslipidemia may happen. Overweight and obesity in children and adolescents is a public health problem worldwide. The prevalence of overweight and obesity in China youth has increased to 13.1% and 7.5% in 2006–2010 from 1.8% and 0.4% in 1981- 1985 [As far back to 1980’s, mass screening of blood cholesterol as routine health care of children was discussed in several industrialized countries  and someth percentile while glycohemoglobin significantly increased at the 99th percentile [2 for boys and 16.3 kg/m2 for girls [Some researches about the cut-off point to predict CVD risk factors were done. Skinner, et al. found that TC increased significantly at the 80rcentile . The resor girls .To update, there was no study aiming to determine the cut-off value to predict the risk factors of CVD in Chinese adolescents. Hence our study intended to illustrate the characteristics of parameters of glycolipid metabolism in obese and overweight adolescents and analyze relevant factors thus to find the best out-off value of BMI-SDS to predict dyslipidemain Chinese youth.Fourteen elementary and junior high schools in Shanghai’s two districts were chosen by using cluster sampling. Questionnaires investigating body weight and height and physical activities were distributed to all the 9 to 15 years old adolescents. The subjects’ body weight and height were measured by trained health care teachers. Totally 10,423 adolescents were screened, among which 1,320(12.66%) were overweight, 699(6.70%) were obese and 8404 have normal weight. Depending on BMI, subjects were divided into normal weight group and high BMI group according to the Chinese BMI-for-age/gender reference . In highObesity was defined on the basis of BMI above the 95th percentile for age and gender on the BMI percentile charts for Chinese children and adolescents and overweight above the 85th percentile .Team members of this study were trained before research. Body weight and height were obtained using Seca stadiometer and electronic scale, with the participants wearing light underwear and barefoot. Waist circumference was measured at the narrowest point between the lower border of the rib cage and the iliac crest. Physical examination was performed to evaluate the participants’ health status. Non alcoholic fatty liver disease (NAFLD) was identified by ultrasonography and recorded as absent, mild, moderate and severe . The exa2 (m2). As BMI and WC change with age and sex, BMI standard deviation score (BMI-SDS) and WC standard deviation score (WC-SDS)were calculated by LMS (L(lambda), M (mu), S (sigma)) method [Body mass index (BMI) was calculated according to the equation: BMI = body weight (Kg)/ height) method  for each) method ,20.Blood samples were taken from the participants after at least 8-hour fast. Total cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), apolipoprotein A1, Apolipoprotein B, glucose, glycatedhemoglobin ratio, serum lipoprotein a, insulin, peptide C were tested by standard methods using a Hitachi 7180 analyzer . The coefficients of variation (CV) of intra-assay were less than 8% and inter-assay less than 10% for all the items. Electrochemical Luminescence was used to measure insulin and peptide C by Roche E601 analyzer. The CV of intra-assay and intermediate precision were ≤1.50% and ≤4.90% for insulin, ≤4.60% and ≤5.00% for peptide C.According to the IDF criteria hypertriglyceridemia is defined as ≥150 mg/dL (1.70 mmol/L) for triacylglycerol, low HDL-C < 40 mg/dL (1.03 mmol/L) and hyperglycaemia as fasting glucose ≥ 5.6 0 mmol/L . The cutSSPS 19.0 was used to perform statistical analysis. For normally-distributed variables, mean ± standard deviation of data was given. For non-normally distributed data, median, interquartile range was given. Student-Newman-Keuls test was used to compare the means among groups while data met the criteria. Otherwise medians were compared by Kruskal-Wallis Htest. Chi-square test was used to compare the prevalence of fatty liver, high TC and other dyslipidemia among different groups. Simple correlations were done to calculate the correlation indices between BMI-SDS percentiles and the variables. Receiver operating characteristic (ROC) curves of BMI-SDS to predict hypertriglyceridemia and low HDL-C and high LDL-C were made. The best cut-off values were determined while the Youden index was maximum .Five hundred and thirty-eight adolescents participated in this research, among which 283 had normal body weight, 115 were overweight and 140 were obese. Table The prevalence of dyslipidemia and NAFLD among three groups was shown in Table The concentrations of lipids and glucose in boys were shown in Figure The correlations between BMI percentiles and the glycolipid profiles were shown in Table The ROC curve reflecting high level of TC, TG, LDL-C and low level of HDL-C from BMI-SDS in boys was shown in Table The ROC curve reflecting dyslipidemia  from BMI-SDS in boys was shown in Figure Odds ratios for BMI-SDS and BMI in relation to the prevalence of abnormal lipid profiles were presented in Table The AUCs of BMI-SDS and BMI in girls for predicting dyslipidemia were close to 0.5 with p values higher than 0.05. Thus the cut-off point in girls couldn’t be determined.vs. 8.6%, 6.13% vs. 7.5%, 2.97% vs. 6.6% respectively). It may due to the racial difference, different dietary pattern and age coverage.The results of our study showed that obese and even overweight children had dyslipidemia , especially in boys. Thus the reference of Chinese BMI cut-off for overweight and obese can effectively reflect the risk factors of cardiovascular diseases, relatively poor in girls. The concentrations of LDL-C and TG in normal, overweight and obese youths were slightly lower than the results of a study which took WHO cut-off values, while the level of HDL-C was slightly higher . CompareThe concentration of TC in our study was consistent with the results of screening study performed among children and adolescents in America . While tThe proportion of children with hypertriglyceridemia and low HDL-C in obese group were about 10.70% and 6.43%, which were lower than 23% and 23.2% reported by de Onis . BesidesThe best cut-off of BMI-SDS to dyslipidemia in boys was 1.22, which fell in the range between the values of diagnose of overweight and obesity. This value covers not only obese children but also about two-thirds overweight ones, which is in line with the aggressive stance on lipids screening among overweight and obesity youths recommend by AAP . The poiCompared to the cutoff points of dyslipidemia or high TG level, the predictive cut-off value of BMI-SDS for low HDL in boys was 2.19, which was much higher than those for high TG and LDL. It also meant something. It was compliance with the research carried out in Italian youths . In 1080Interestingly, the glycolipid profiles in girls with different BMI did not differ significantly. This phenomenon was not reported by other researchers. One reason was some study did not analyze the gender differences . AlthougThough the cut-off value of BMI-SDS and BMI for predicting dyslipidemia in boys was calculated in this study, it should be careful to be applied into practice. The population sample was located in Shanghai urban area, it may not be generalized to other population. The sample size of our study was relatively small, larger studies are needed to confirm or refute our findings.The results of this study showed that overweight and obese youths had high levels of TC, TG, LDL, low HDL level and reduced insulin sensitivity. BMI-SDS can predict dyslipidemia at the value of 1.22. Therefore, besides the attention to obese youths, more focus should be casted on overweight ones.BMI-SDS: Body mass index standard deviation score; LDL-C: Low-density lipoprotein cholesterol; HDL-C: High density lipoprotein cholesterol; TG: Triglyceride; TC: Total cholesterol; CVD: Cardiovascular diseases; AAP: American Academy of Pediatrics; NAFLD: Nonalcoholic fatty liver disease; CV: Coefficients of variation; HbA1C%: Hemoglobin A1c; C-P: Peptide C; HOMA-IR: Homeostasis model assessment of insulin resistance; OR: Odds ratio; CI: Confidence interval; ROC: Receiver operating characteristic; AUC: Area under the ROC curve.The authors declare that they have no competing interests.YMP and ZYZ were responsible for the initial conception and design of the study. CDG, QLW, ZC, DZ carried out the survey.CDG contributed to the statistical analysis and interpretation data and wrote the first draft of the paper. All authors contributed to the critically revision of the article and approved the final published version to be published.


The response time of the chip was also experimentally verified to be within 5 seconds from RH 36% to RH 64%. As a consequence, the implemented humidity SSoC paves the way toward the an ultra-small sensor system for various applications.A fully integrated humidity sensor chip was designed, implemented, and tested. Utilizing the micro-stamping technology, the pseudo-3D sensor system-on-chip (SSoC) architecture can be implemented by stacking sensing materials directly on the top of a CMOS-fabricated chip. The fabricated sensor system-on-chip (2.28 mm × 2.48 mm) integrated a humidity sensor, an interface circuit, a digital controller, and an On-Off Keying (OOK) wireless transceiver. With low power consumption,  To build ubiquitous sensor networks, moreover, the small system form factor should be addressed to seamlessly integrate the sensor system into environments. To match the above requirements, monolithic sensor architecture has been proposed and implemented by micro-electro-mechanical system (MEMS) technology for years [i.e., sensing area is much larger than the circuit area. As a consequence, novel sensor chip architectures need to be developed.With the advancement of communication and information technologies, sensor networks have become an emerging technology to be developed for the next generation . This teor years ,4. Howev4, CO and CO2 environment, and good temperature stability of 7.85 × 10−10 A/°C [Among the various sensing quantities sought in different applications, humidity is one of the most important factors –7. As a −10 A/°C . In thisWith the circuits fabricated by a CMOS process, specifically the Fe-Al-polyaniline humidity sensing material is micro-stamped on the top of CMOS circuits to form the pseudo three-dimensional (3D) sensor chip architecture. 3D integration is one of the methods to integrate heterogeneous layers with interconnecting metal lines to improve performance or create new functions of chips . With th2.2.1.3+ and Al3+ with specific concentration, can affect the selectivity and resistance of the blends. Following stirring within 200 mL 10% NaOH solution and precipitation, polyaniline blends can be obtained. Subsequently, the precipitate is washed with distilled water to reduce the acidity [To fabricate the sensing material, a solution-based process has been developed . In brie acidity ,15. The 2+ +H2O → NH + H3O+, is stimulated as the sensing film is exposed to humidity. As a result, the resistance of the polyaniline blends decrease. This reaction can be triggered in room temperature. The resistance change of polyaniline blends introduced by the reaction between low and high relative humidity (35% to 70% RH) is three orders [The fundamental sensing mechanism of the polyaniline film has been investigated in previous works . In briee orders . The int2.2.6 to 108 [To the analog front-end (AFE) circuit, the humidity sensing device plays as a variable resistance in the voltage dividing circuit, as shown in 6 to 108 , the vol6 to 108 .i.e., a cell phone or laptop, for further data analysis and saving. In addition, the wireless circuit cannot only transmit data but also receive command codes to wake up this SSoC from standby mode to active operation mode. Moreover, this chip also includes a built-in junction-based temperature sensor with good linearity. This temperature sensor shares the same chopper DDA with the humidity sensor by a multiplexer to reduce the circuit power consumption and complexity. Although the developed polyaniline blends have good temperature stability (7.85 × 10−10 A/°C) [After the sensing is amplified, the sensing signal is then translated into digital data by a 10-bit successive approximation register analog-to-digital converter SAR ADC with low power consumption. With the help of a built-in digital signal processor (8051-based), this digital data is packaged into RS232 format to be sent by OOK wireless circuits (TX/RX) to an external host, 10 A/°C) , this te2.3.To implement the proposed system architecture, the Taiwan Semiconductor Manufacturing Company (TSMC) 0.35 μm two-polysilicon-four-metal (2P4M) CMOS process is employed to implement the humidity sensor system. Previously designed analog and digital circuits are implemented following a standard CMOS process flow. The top metal layer  of the chip is used as the interdigitated electrodes. These interdigitated electrodes are directly connected to the AFE input by vias. It should be noted that the position of the intedigitated electrodes is on the top of the chopper DDA and OOK TX/RX to form the proposed pseudo-3D architecture. On the top of interdigitated electrodes, a layer of gold is e-beam evaporated to form a good contact to the sensing material . Then th2.4.The humidity sensing experiment was designed and used to determine the performance of the developed heterogeneous humidity sensing SoC. The testing board module can be seen in 3.2. The performance table shows the power consumption of the circuit system is around 730 μW without RF operation. By considering the sensing device and the off-chip resistor operation, the total power consumption is around 750 μW. The region marked by a blue line is the place for stamping the sensing material. After stamping the sensing material, the chip is wire-bonded and ready for test.The fabricated sensor chip photo and electrical performance are shown in i.e., from 32% to 66% in controlled chamber. The experimental results are shown in To examine if the sensing material is functional on the top of the interdigitated electrodes on the fabricated chip, the humidity SSoC was placed into a controlled chamber. With gating the injection of different relative humidity into the chamber, the voltage signal at the output of AFE was measured by Keithley 2400 source-meter. The experimental date of a single humidity injection can be shown in 4.2). With low power consumption, i.e., 750 μW without RF operation, experimental results have shown that the humidity sensing SoC had a fast response time of about 5 seconds and high sensitivity of about 30 mV/% at room temperature. In addition, the SSoC integrates a polymer-based humidity sensing device, AFE, SAR ADC, 8051-based microcontroller, and OOK wireless TX/RX on a single chip. Therefore, this work demonstrates not only a practical humidity sensor system-on-chip, but also a novel method to implement sensor systems for applications in sensor network technologies.In this work, a fully-integrated humidity sensor system-on-chip (SSoC) is demonstrated. The SSoC is fabricated by the TSMC 0.35 2P4M CMOS process. Implemented by a micro-stamping technique to stack the sensing device on the top of CMOS circuits, the SSoC can be formed in a pseudo 3D architecture to obtain an ultra-small form factor (5.65 mm


Melastoma malabathricum L. (Melastomaceae) is a small shrub with various medicinal uses. The present study was carried out to determine the hepatoprotective activity of methanol extract of M. malabathricum leaves (MEMM) against the paracetamol-induced liver toxicity in rats model.The respective chemicals and herbal solutions ) were administered orally to rats once everyday for 7 days followed by the hepatotoxicity assay. The blood samples and livers were collected and subjected to biochemical and microscopical analysis. Prior to the hepatoprotective study, MEMM was subjected to determination of the total phenolic content (TPC) and the antioxidant properties using several standard assays .MEMM exerted significant (p < 0.05) and high antioxidant activity in which high TPC was recorded; while in the hepatotoxicity study, the extract exhibited significant hepatoprotective effects against the paracetamol-induced hepatotoxic model. The results observed for serum liver enzymes  as well as the microscopic observations and microscopic scoring supported the hepatoprotective potential of MEMM. The phytochemical and HPLC analysis of MEMM demonstrated the presence of flavonoids as its major constituents.The MEMM-induced hepatoprotective activity could be allied partly to its antioxidant activity and the presence of flavonoids. Liver is the largest organ in the human body and is a key organ of metabolism ,2. DespiMelastoma malabathricum L., which is locally known as “Senduduk”. This small shrub belongs to the family Melastomaceae and is native to tropical and temperate Asia, including Malaysia, and the Pacific Islands. Despite being a well-known herb in Malaysia wherein its leaves, shoots and roots are prepared in various ways to treat various types of diseases , attempts to scientifically investigate and confirm those claims are however lacking [M. malabathricum have been prepared and tested using a variety of in vitro and in vivo test models. The plant has been reported to possess various types of pharmacological activities  at different doses or concentrations [Interestingly, in line with increase in patients attempt to use complementary and alternative medicines, particularly herbal/plant-based therapies, to cure various diseases, efforts are increasingly being carried out by scientists to investigate the hepatoprotective potential of various medicinal plants. One of the medicinal plants that have been widely used in the Malay traditional medicine is  lacking . Scientitrations .M. malabathricum[″-O-gallate, malabathrin-A, -B, -C, -D, -E and -F, 1,4,6-tri-O-galloyl-β-D-glucoside, 1,2,4,6-tetra-O-galloyl-β-D-glucoside, strictinin, casuarictin, pedunculagin, nobotanin-B, -D, -G, -H and -J, pterocarinin C, new complex tannins in which an ellagitannin and a flavan-3-ol are bound by a C-glycosidic linkage belonging to type II + tannins, casuarinin, (−)-epicatechin gallate, (−)-epicatechin, stachyurin, procyanidin-B2 and -B5, stenophyllanins A and B, alienanin B, and brevifolincarboxylic acid; ii) methanol extract – ursolic acid, 2-hydroxyursolic acid and asiatic acid, as well as glycerol-1,2-dilinolenyl-3-O-β-Dgalactopyranoside and glycerol 1,2-dilinolenyl-3-O- -β-D-galactopyranoside, 2,5,6-trihydroxynaphtoic carbonic acid, methyl-2,5,6-trihydroxynaphtalene carbonate, flavonol glycoside derivative, quercitrin and kaempferol-3-O--β-glucoside; iii) hexane fraction of methanol extract – β-sitosterol, α-amyrin, uvaol, quercetin, quercitrin, rutin, and sitosterol-3-O-β-D-glucopyranoside; iv) 90% aqueous methanolic extract – ursolic acid, 2α-hydroxyursolic acid and asiatic acid, β-sitosterol 3- O-β-Dglucopyranoside, glycerol 1,2-dilinolenyl-3-O-β-D-galactopyranoside and glycerol 1,2-dilinolenyl-3-O--β-D-galactopyranoside; v) ethyl acetate extract – 2,5,6-trihydroxynaphtoic carbonic acid, methyl-2,5,6-trihydroxynaphtalene carbonate, and flavonol glycoside derivative, quercetin and quercitrin; and vi) hexane extract – 2,5,6-trihydroxynaphtoic carbonic acid, methyl-2,5,6-trihydroxynaphtalene carbonate, and flavonol glycoside derivative, α-amyrin, patriscabatrine and auranamide.Various phytochemical constituents have been isolated and identified from various parts and extracts of bathricum. FocusinM. malabathricum and the reports relating, at least, the anti-inflammatory and antioxidant activities to the hepatoprotective mechanism [M. malabathricum. It is postulated that M. malabathricum leaves extract, which possesses the anti-inflammatory activities, will also exert antioxidant and hepatoprotective activities. Therefore, the aim of the present study was to determine the hepatoprotective activity of methanol extract of M. malabathricum (MEMM) using the paracetamol-induced liver damage in rats as the animal model. In addition, the antioxidant activity, phytochemical content and HPLC profile of MEMM were also verified to support the hepatoprotective potential of the extract.Based on the previously demonstrated anti-inflammatory activity of echanism -11, the M. malabathricum were continuously collected from their natural habitat in Serdang, Selangor, Malaysia between September 2011-Jun 2012 and identified by comparison with specimens available at the Herbarium of the Laboratory of Natural Products, IBS, UPM, Serdang, Selangor, Malaysia. Sample of the plant was also deposited at the same herbarium .The leaves of The plant extraction procedure was carried out according to the previously described method . BrieflyParacetamol, silymarin, nitroblue tetrazolium salt (NBT), phenazine methosulfate (PMS), 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), Trolox , a water-soluble analog of vitamin E, and β-phycoerithrin, were purchased from Sigma-Aldrich Co.  and used in the present study. All other chemicals and reagents used were of analytical grade.ad libitum from the beginning of the experiments. The study protocol of the present study was approved by the Animal House and Use Committee, Faculty of Medicine and Health Sciences, UPM . The rats were handled in accordance with current UPM guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals. All experiments were conducted between 09.30 and 18.30 h to minimize the effects of environmental changes.Male Sprague Dawley rats  and male ICR mice  were obtained from the Veterinary Animal Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM), Malaysia and kept under room temperature  in the Animal Holding Unit (UPM). They were supplied with food and water In an attempt to determine the antioxidant potential of MEMM, the total phenolic content (TPC), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, superoxide anion radical scavenging and oxygen radical absorbance capacity (ORAC) of MEMM was determined according to the respective method of -16 but wMeasurement of superoxide anion radicals scavenging activity of MEMM was based on the method described by Liu et al. . Superox0 was the absorbance of the control (L-ascorbic acid), and A1 was the absorbance in the presence of MEMM or standards.where AThe microplate fluorescence reader with an excitation wavelength of 540 nm and an emission wavelength of 565 nm was used . The 75 2O) ad libitum under standard laboratory conditions. After 48 hours, each group of rats  received the respective dose of chemical (10% DMSO) or herbal  solutions orally once daily for 7 consecutive days. The doses of MEMM were selected based on the single dose (5000 mg/kg) acute toxicity study performed in the previous studies. The MEMM did not cause any signs of toxicity at the dose of 5000 mg/kg according to the results shown by all the blood parameters and serum biochemical analysis. However, this dose was found to cause significant reduction of the rats’ body weight. Therefore, the doses range of MEMM  used in the present study was selected based on the 100, 20 and 10 folds reduction of the dose 5000 mg/kg used in the toxicity study. The oral administration of paracetamol was performed 3 hours after the administration of the last chemical or herbal solutions on the 7th day. As for Group I, the rats were pretreated with 10% DMSO for 7 consecutive days followed by the administration of 10% DMSO on the 7th day. The animals were divided into 6 groups and treated with chemical or herbal  solutions as described below.The hepatoprotective activity of MEMM was determined using paracetamol-induced hepatotoxicity model, of which rats were used . The ani• Group I received 10% DMSO + 10% DMSO.• Group II received 10% DMSO + paracetamol.• Group III received 200 mg/kg silymarin + paracetamol• Pre-treatment groups;• Group IV received 50 mg/kg MEMM + paracetamol,• Group V received 250 mg/kg MEMM + paracetamol and;• Group VI received 500 mg/kg MEMM + paracetamol.Forty eight (48) hours after the procedure induced hepatic injury, the animals were anesthetized using diethyl ether and the blood was withdrawn for biochemical analysis. The animals were then sacrificed by cervical dislocation and the liver was removed for histopathological studies according to previously published method .Biochemical parameters were assayed according to the standard methods as described by Yahya et al. . The alaThe liver tissue was dissected and processed according to the method of El-Beshbishy et al.  but withThe phytochemical screening of MEMM was performed according to the standard screening tests as adopted by Zakaria et al. . The resSamples were soaked in chloroform followed by addition of ammoniacal chloroform. The mixture was then treated with sulphuric acid 10% and further tested with Mayer’s reagent. Formation of white precipitates indicates the presence of alkaloids.The methanolic extract of samples were dissolved in ether and shaken in 10% ammonia solution. Formation of yellow colour in ammonia layer indicates the presence of flavonoids.The methanolic extract of sample was analysed using Liebermann-Buchard reagent. The extract was mixed with few drops of acetic anhydride, boiled and cooled. Concentrated sulphuric acid was then added from the sides of the test tube and observed for the formation of a brown ring at the junction of two layers. Green coloration of the upper layer and the formation of deep red color in the lower layer would indicate a positive test for steroids and triterpenoids, respectively.The methanolic extract of samples was mixed with 1% ferric solution. Formation of blue black colour indicates the presence of hydrolysable tannins, while brownish-green indicates that of condensed tannins.The methanolic extract of samples was mixed with distilled water in a test tube. Formation of stable froth for at least 15 minutes indicates the presence of saponins.18 column with dimensions 250 x 4.60 mm using a one-step linear gradient. The solvents were (A) 0.1% aqueous formic acid and (B) acetonitrile and the elution system was as follows: Initial conditions were 85% A and 15% B with a linear gradient reaching 25% B at t = 12 min. This was maintained for 10 min after which the programme returned to the initial solvent composition at t = 25 min and continued for 10 min. The flow rate used was 1.0 ml/min and the injection volume was 10 μl. The HPLC was set at 254 and 336 nm.The HPLC profiling of MEMM was carried out according to the methods described by Zakaria et al.  with sliIn an attempt to identify the bioactive compound(s) present in MEMM, several standard flavonoids  were injected into the HPLC system either alone or in combination with MEMM. The selection of flavonoid-based compounds was based on the range of wavelength of each peak obtained earlier.P < 0.05 as the limit of significance.Data obtained are presented in mean ± standard error of mean (SEM). The data were analysed using the one-way analysis of variance (ANOVA) and the differences between the groups were determined using the Dunnet post hoc test with The results of antioxidant potential of MEMM assessed using various antioxidant assays are shown in Table The administration of paracetamol following pretreatment with 10% DMSO (Group II) significantly (p < 0.05) increased the liver weight of rats when compared to the normal group (Group I) Table . In contThe liver collected was subjected to macroscopic and microscopic investigations to detect irregularities or abnormalities of the structure due to paracetamol. Macroscopic analysis of the liver intoxicated by paracetamol (Group II) demonstrated major brown color changes of the liver lobes Figure B in compMicroscopically, the non-paracetamol-intoxicated group  exhibited normal lobular architecture wherein the cytoplasm is well preserved with the normal hepatic cells of which a well defined sinusoids line and nucleus around the perivenular area are observed Figure A. In comOral treatment with paracetamol following the 10% DMSO pretreatment (Group II) significantly (P < 0.05) elevated the ALT, AST and ALP serum marker level as compared to the normal group  Table . InteresThe phytochemical constituents of the dried leaves and MEMM are shown in Table max values in the region of 234.9, 254.3-367.2, 204.9-348.2 and 255.5-369.4 nm, respectively  presence in the extract. Interestingly, the MEMM was found to contain quercetin, quercitrin and rutin Figure C.Paracetamol-induced liver injury model is a conventional model used to investigate the hepatoprotective activity of extracts/compounds . In the 4 is transformed by the cytochrome P450 system to produce highly reactive trichloromethyl free radicals whereas upon the administration of high dose of paracetamol, the sulfation and glucuronodation routes become saturated causing the high percentage of paracetamol molecules to be oxidized to highly reactive N-acetyl-p-benzoquinone imine (NAPQI) [Various mechanisms may be linked to the damage of the liver by different toxins. For examples, the CCl (NAPQI) . NAPQI,  (NAPQI) ,22 that  (NAPQI) . NAPQI a (NAPQI) . Moreove (NAPQI) . The dep (NAPQI)  that is  (NAPQI) ,28. More (NAPQI) ,28.The present study demonstrated the ability of MEMM to exert hepatoprotective activity and supported our earlier hypothesis that the extract possessed anti-inflammatory, antioxidant activities and also hepatoprotective effects. The hepatoprotective potential of MEMM was further supported by the ability of the extract to reduce the serum liver enzymes (ALT and AST) level and the liver/body weight ratio, and the histopathological findings. However, through this study also the consumption of MEMM for 7 days was found to reduce the body weight of rats. This finding is in accordance with previous studies by Shimoda et al.  and AuviLately, much interest has been given towards the possible involvement of oxidative stress in the initiation and/or progression of paracetamol-induced hepatotoxicity. Reactive oxygen and nitrogen intermediates, generated by hepatic paranchymal and non-paranchymal cells are thought to play important roles that lead to paracetamol-induced injury -36. TherOther than the oxidation processes, the inflammatory processes have been thought to be intimately involved in the chemical-induced hepatotoxic processes . The infM. malabathricum extracted using various organic solvents extracts have been reviewed elsewhere [max falling in the region of 234.9, 254.3-367.2, 204.9-348.2 and 255.5-369.4 nm. These peaks may represent flavonoid-types of bioactive compounds  . The UV–vis spectra of flavonoids consist of two absorbance bands labeled as band A and band B [Phytochemical constituents have been known to play important role in determining the pharmacological potentials of various medicinal plants. Various types of bioactive compounds have been isolated, identified and scientifically proven to possess certain types of pharmacological activities from various types of plants. In term of the MEMM, flavonoids, tannins, saponins and steroids have been identified in the extract, which has also been reported to possess high TPC value . The prelsewhere . In termlsewhere ,56, antilsewhere  and hepalsewhere  activitilsewhere ; saponinlsewhere  and antilsewhere . Based od band B . For flad band B . Based oThe MEMM exhibited hepatoprotection against paracetamol-induced liver injury model, which could be, partly, attributed to its antioxidant activity and, linked to the presence and synergistic action of flavonoids, tannins and saponins. Further studies are warranted and are being planned to determine the possible hepatoprotective mechanism(s) involved and, to isolate and identified the responsible bioactive compounds.The authors declare that they have no competing interest.SSM, MFFK, and FY carried out the animal studies, biochemical analysis and drafted the manuscript. NDM, MSS and KFJ carried out the antioxidant studies, phytochemical screening, HPLC analysis and helped in the preparation of manuscript. NM involved in the macroscopic and microscopic analysis and helped to draft the manuscript. SMC involved in the statistical analysis and and helped to draft the manuscript. DS and MT helped in the phytochemical and HPLC works. ZAZ conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/13/326/prepub


Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers.  The term “quorum sensing”, introduced by Peter Greenberg in 1994 Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbelli(Z)-3-aminoundec-2-en-4-one (Ea-C8-CAI-1)] luxR transcript luxRqrr2-4 transcription by LuxR luxO and repression of luxO translation by Qrr sRNAs luxMN translation by Qrr sRNAs Several feedback loops are known to regulate the content of LuxR in the cells. These involve autorepression of V. harveyi produces three AIs but channels all information into a single signaling cascade. Moreover, we have previously shown that extracellular concentrations of AIs correlate with the degree of cell-to-cell variance in the expression of bioluminescence 6 cells*mL−1, OD600 = 0.001) to the stationary growth phase . It should be noted here that, in previous studies, the expression of AI-regulated genes has been analyzed predominantly by studying their responses to exogenously provided AIs luxR transcript levels and diverse AI-regulated processes. Our data suggest a model in which the precise composition of the AIs present in certain growth phases, rather than the cell density per se, is the more important influence on AI-regulated gene expression. This model is supported by in vitro phosphorylation studies.In spite of detailed knowledge of the complex signaling cascade, it is still unclear why V. harveyi strains listed in −1). Overnight cultures were diluted 5,000-fold into fresh AB medium and grown for a further 20 h. Samples were taken every hour, and cells were removed by centrifugation at 5,000× g for 15 min. The culture fluids were then filtered (0.20 µm) and stored at −20°C or used immediately. To measure the cell density of a V. harveyi culture the optical density at 600 nm was determined for values larger than 0.01. For cultures with an OD600<0.01 the number of colony-forming units was determined directly, and the optical density was calculated (OD600 = 1 corresponds to 109 cells*mL−1).The Escherichia coli strains listed in −1) or chloramphenicol (33 µg*mL−1).luxN and luxQ in E. coli TKR2000 each gene was inserted into plasmid pKK223-3, in which expression is under control of the tac promoter. To use the natural Shine Dalgarno box of kdpD, plasmid pPV5-1  or via a two-amino acid linker . Inside-out membrane vesicles were prepared as described E. coli MDAI-2 transformed with the plasmid pGEX_LuxP as described before E. coli JM109 transformed with plasmid pQE30LuxU-6His LuxP was produced in and purified from Protein concentrations were determined by the method of Peterson −1 causing a back-pressure of 770 bar. Detection was performed at 195 nm with a scan rate of 20 Hz. The analysis time for each injection was set to 3 min, and all sample constituents were eluted from the column. The retention time for HAI-1 (0.579 min) and the UV-Vis spectra of the peak provided the criteria for identification of the compound and assessment of its purity. A standard solution of HAI-1 was used for calibration and quantification of the analyte.HAI-1 was quantified by UPLC using an Acquity UPLC System with a 2996 PDA detector controlled by Empower software (Waters). The system was equipped with an Acquity 2.1×100 mm BEH C18 column packed with 1.7-µm particles (Waters), which was maintained at a constant temperature of 60°C. The Sample Manager was kept at 27°C. Aliquots (5 µl) of sample were injected via a partial loop with needle overfill, and all samples were analyzed three times. Water (Biosolve) containing 3% acetonitrile (Biosolve) served as the mobile phase, and isocratic elution was applied at a flow rate of 0.9 mL*min−1) was added, and the mixture was incubated at 30°C for 30 min. The LuxP-AI-2 complex was then separated from the culture fluid by centrifugation of the sample through a NMWL filter (Millipore) with a 30,000-dalton cut-off, so that the protein-AI-2 complex was retained on the filter. To dissociate the complex, the membrane was washed in water, and the extract was kept at 50°C for 10 min and filtered again. The filtrate containing the AI-2 molecules was subsequently used in a bioluminescence assay with V. harveyi MM77 as reporter strain. To obtain a calibration curve, standard solutions of synthetic AI-2 (0 to 50 μM) were tested in the reporter assay with V. harveyi MM77 (see below). The fitted lines for the HAI-1 and AI-2 concentrations presented in To determine the concentration of AI-2 in culture fluids, LuxP-GST ]. In parallel, CAI-1 was analyzed by GC-TOF-MS. Metabolites in culture fluids prepared as described above were chemically modified by sequential methoxyamination and trimethylsilylation, as described earlier http://www.leco.de). Selective ion traces and peak heights were extracted from the NetCDF CHROMATOF export, and processed using the TagFinder software http://gmd.mpimp-golm.mpg.dehttp://gmd.mpimp-golm.mpg.de/search.aspx using the “A” identifier code (see legend to CAI-1 levels in cell-free cultures fluids of wild type in vitro using S-adenosyl-homocysteine (Sigma-Aldrich) and the enzymes LuxS and Pfs, followed by purification over boric acid resin E. coli JM109 transformed with plasmid pQE30LuxS-6His or pQE30Pfs-6His, respectively, and purified as described before in vitro phosphorylation experiments with LuxN/LuxQ and LuxU.HAI-1 (D- and L-isomers) was purchased from the University of Nottingham and dissolved in a minimal volume of acetonitrile [10% (v/v)], diluted with water to a concentration of 100 mM and stored at −20°C. DPD, the precursor of AI-2, was synthesized V. harveyi strains was determined in microtiter plates in a Centro LB960 (Berthold Technology) for 0.1 s, and data are reported as light units (LU) or relative light units (RLU) [counts*s−1] per OD600 unit. All measured data were below the saturation range of the instrument (2.2*106 LU). To determine the dose-dependent effect of HAI-1 or AI-2, strain MM77 (luxM::Tn5 luxS::Cmr) was used as reporter. Overnight cultures of strain MM77 were diluted 1∶100 in AB medium containing culture fluids [50% (v/v)] or various concentrations of synthetic HAI-1 and AI-2. Cells were grown until the mid-exponential growth phase and analyzed as described above.Luminescence produced by V. harveyi strains was measured by incubating hide powder azure (Sigma-Aldrich) in phosphate-buffered saline  with cell-free culture fluids at 37°C. The reaction was stopped with trichloracetic acid [6.7% (v/v)] after 2 h, and the absorbance at 600 nm was measured Exoproteolytic activity of V. harveyi strains BB120 and JMH634 were cultivated as described above. Samples were withdrawn, and RNA was isolated as described before luxA, luxR, vhpA, vopN, vscP and recA. Duplicate samples from three independent biological experiments were used, and the CT value (cycle threshold) was determined after 40 cycles using the iQ software (Biorad). Values were normalized with reference to recA and relative changes in transcript levels were calculated using the comparative CT method Phosphorylation reactions were performed in phosphorylation buffer  glycerol, 500 mM KCl, 2 mM DTT) at 25°C. The sensor kinases LuxQ and LuxN were tested as full-length membrane integrated proteins in inverted membrane vesicles. To incorporate LuxP into LuxQ-bearing membrane vesicles, vesicles were subjected to three cycles of freezing and thawing.−1 (LuxQ) or 5 mg*mL−1 (LuxN) membrane proteins, and 0.36 mg*mL−1 purified LuxU. LuxP was added at a concentration of 0.96 mg*mL−1. For experiments involving both kinases, the concentration of each kinase was halved. The reaction was started by addition of radiolabeled Mg2+-ATP, typically 100 μM [γ-32P] ATP  and 110 μM MgCl2. At various times thereafter, the reaction was terminated by the addition of SDS loading buffer 32P] ATP were used to generate a calibration curve for quantification of the signal intensities of phosphorylated proteins using ImageQuant software . All enzymatic activities were calculated as mean values of at least three independent experiments. The gels shown are representative of each set of experiments.A typical reaction mixture for a phosphorylation assay  contained 7.5 mg*mLg, 15 min, 4°C), and ATP was removed by gel filtration . Dephosphorylation of phosphorylated LuxU (0.18 mg*mL−1) was then initiated by the addition of 110 μM MgCl2 and membrane vesicles containing LuxQ (3.75 mg*mL−1). Phosphorylated LuxU was quantified as described above.For dephosphorylation assays LuxU was first phosphorylated using LuxN. In this case, the reaction mixture contained twice the usual amounts of LuxN and LuxU. After 10 min of incubation, membrane vesicles were removed by centrifugation  grown in liquid AB medium at regular intervals .The extracellular concentrations of the three AIs were determined in a wild type population of ntervals . To starV. harveyi either ceases to produce this AI at a certain point and/or the bacterium has other ways of reducing the number of AI-2 molecules present in the medium. The time course for HAI-1 productivity per cell corresponds to the typical threshold-mediated regulation in quorum sensing.To estimate the productivity of the population, the measured concentrations of HAI-1 and AI-2 were normalized to the corresponding cell density . RemarkaV. cholerae MM920 reporter strain V. harveyi JHM626 was used as reporter strain, which revealed comparable results with a basal CAI-1 activity during exponential growth and a high activity within the stationary phase (data not shown). These results were supported by GC-TOF-MS profiling , which is in agreement with earlier reports The  rapidly . This reV. harveyi culture. Since AI-dependent induction of a gene encoding a putative exoprotease has only been described at the transcriptional level luxO-Cmr). The quorum sensing negative mutant JAF548 (luxO-D47E), as well as the mutant MM77  which is unable to produce HAI-1 or AI-2, exhibited very low activities. Proteolytic activity could be restored in mutant MM77 by adding both HAI-1 and AI-2 at physiological concentrations , but was insensitive to phenylmethylsulfonyl fluoride (PMSF) (In the next experiment we determined the time course of the AI-dependent induction of exoproteolytic activity in cell-free culture fluids from a growing trations . These de (PMSF) .V. harveyi population indicated that exoproteolytic activity was absent during the first 10 h of cultivation , significantly earlier than in the untreated wild type population (at 10 h) . AlthougIn summary, induction of the exoprotease is temporally decoupled from the onset of bioluminescence, despite the fact that the corresponding genes are primarily under the control of the same signaling cascade. This notion supports the idea that different blends of AIs drive different outputs.V. harveyi mutant MM77  after adding different concentrations and mixtures of AI-2 and HAI-1. Induction of bioluminescence showed a linear dependence on AI-2 concentration over the range from 0.1 to 5 μM . The L-HAI-1 isomer caused no significant induction (data not shown), which is in agreement with the known stereospecificity of V. harveyi HAI-1 luxS/cqsA mutant, which produces only HAI-1 luxM/luxS mutants KM413 When we tested D-HAI-1, we also found that bioluminescence was induced, albeit with much reduced efficacy. The level of bioluminescence induced by D-HAI-1 was only 0.06% of that observed following the addition of the same concentration (5 μM) of AI-2 . The funV. harveyi  on its own, the simultaneous presence of HAI-1, which has only a minor effect by itself , significantly increases the level of bioluminescence observed .We therefore tested the effects of HAI-1 and AI-2, applied in different molar ratios, on the induction of bioluminescence . ImportaThen we tested the dose-dependent effect of AIs on the induction of the exoprotease. An increase in the AI-2 concentration led to a concomitant increase in the exoproteolytic activity. HAI-1 induced this activity too, but to a much lesser degree , and rose further when HAI-1 appeared in the medium (time point 3). The maximal transcript level was measured at the time when all three AIs were present . The number of transcripts per cell , vhpA (an exoprotease), vopN (an outer membrane protein) and vscP (a putative translocation protein in type III secretion) transcripts all differed in detail . At time point 4 (LuxR level highest), the transcript level of luxA was low. Luciferase is a stable protein, which might explain the transcriptional down-regulation. Nevertheless, the drop in luxA transcript level coincides with the decline in bioluminescence described above -mediated phosphorylation of LuxU as output. The full-length hybrid kinases LuxN and LuxQ (tagged with 6 histidine residues) were expressed in the E. coli strain TKR2000. This strain lacks the F1/Fo-ATPase, and inverted membrane vesicles can be used directly for phosphorylation experiments. Analogously to a biochemical characterization of the HAI-1-recognizing kinase LuxN described earlier −1*mg−1 kinase protein, respectively) (We performed ctively) . Additioctively)  in a conin vivo, we designed an experiment in which the total rate of LuxN and LuxPQ (molar ratio LuxN:LuxQ  = 1∶1) mediated LuxU phosphorylation was assayed in the presence of various combinations of AIs. In so doing, we utilized the physiological concentrations of HAI-1 and AI-2 we had found to be present in a growing wild type culture in vivo . Addition of increasing amounts of AI-2 led to concomitant inhibition of LuxU phosphorylation  that occurs in the exponential growth phase, AI-2 is the major AI in the medium. HAI-1 first becomes detectable and increases in concentration during the late exponential growth phase. Under our growth conditions, CAI-1 was detected at readily measurable levels only in the stationary phase. Since we were unable to determine the molar concentration of CAI-1, the sensitivity of our method is unknown. Therefore, we cannot exclude that physiological relevant low concentrations of CAI-1 are present at earlier growth phases. The delay in secretion of HAI-1 relative to AI-2 is in good agreement with data in a recent report V. harveyi culture are characterized by distinct AI profiles: the early exponential growth phase by low AI-2, the mid-exponential growth phase by high AI-2, the late exponential growth phase and the early stationary phase by a blend of AI-2 and HAI-1, and the later stationary phase by a combination of AI-2, HAI-1 and CAI-1. This classification corresponds well with the staggered expression of bioluminescence and exoproteolytic activity during growth of wild type V. harveyi. Although both phenotypes are dependent on AI-controlled genes and hence on the same signaling cascade, they are not induced simultaneously. The onset of bioluminescence occurs, and light levels reach their maximum, in the exponential growth phase, whereas exoproteolytic activity only sets in after the transition into the stationary phase. These findings are supported by our reporter strain analysis, which indicated that AI-2 is sufficient for induction of bioluminescence and that HAI-1 (even at a low concentration) acts synergistically to enhance light production. In contrast, both HAI-1 and AI-2 were required to induce exoproteolytic activity. Other AI-regulated phenotypes seem also to be affected by different combinations of AIs. Based on our experiments, full repression of vscP and vopN requires only AI-2. Furthermore, the sRNA Qrr4 can be induced by HAI-1 or AI-2, but full induction is attained only when both are present together gfp fusions luxR transcript that encodes the master regulator of the signaling cascade. With each additional AI, levels of luxR mRNA increased. The highest level was measured when all three AIs were present simultaneously. Curiously, no gene is yet known to be regulated by LuxR at this late growth stage.Like luxA and hence for bioluminescence, and for the repression of vopN or vscP. At later growth stages, levels of the luxR transcript increase, and vhpA, which codes for a protease, is induced to a maximal level. In agreement with this, full induction of the exoproteolytic activity requires both HAI-1 and AI-2, and hence a higher copy number of LuxR than does the induction of bioluminescence.LuxR activates and represses more than 100 genes, and both the numbers and relative affinities of its binding sites vary for different genes luxA and vhpA during stationary phase. It is still unclear whether LuxR or AphA The transcriptional analysis raises questions regarding the molecular mechanism of down-regulation of gene expression. For example, significantly decreased transcript levels were determined for in vitro data on receptor-mediated phosphorylation of LuxU, the protein which gathers all information, reveal a very tight correlation between various inputs and outputs. The rate of LuxU phosphorylation decreases linearly with the physiological increase in the AI-2 concentration, and the decrease continues as HAI-1 is added to the mix. Remarkably, the activities of the two histidine kinases LuxN and LuxPQ exhibit some degree of cooperativity, because the effects of AI-2 and HAI-1 were non-additive (in vivo measurements with mutants lacking one or two histidine kinases Our additive . Even atadditive . Furtherin vitro data also complement a comprehensive study on input-output relationships in various feedback-loop mutants in vitro studies have used equal quantities of LuxN and LuxPQ. In future experiments we will integrate the other histidine kinases, and test different ratios of the histidine kinases to take into account the recently described positive luxMN feedback loop and the increased sensitivity to HAI-1 The V. harveyi in the ocean. Bioluminescence might attract organisms of the same species to form aggregates or to settle down on surfaces. V. cholerae is known to possess blue-light photoreceptors V. harveyi also possesses genes encoding proteins with a BLUF domain, a sensor for blue light. Bioluminescence improves the nutrient cycle V. harveyi might use additional AI-2 that is produced by other species. Later, when its population has reached a certain cell density, V. harveyi produces and responds to the species-specific HAI-1. Subsequently, HAI-1 boosts bioluminescence induction. At this growth stage, which coincides with stationary growth and the beginning of biofilm formation Vibrio species V. harveyi ensures that the products of exoproteolysis are made available to its own kind. Unfortunately, no gene is known which is under the control of CAI-1 in the stationary phase. Nonetheless, it is suggested that V. harveyi needs all three AIs to time the onset and duration of certain AI-regulated processes during different stages of growth.The stable succession of different AI-regulated processes might facilitate the proliferation of


Objective(s): Human T-cell lymphotropic virus type-1 is an oncornavirus that causes adult T cell leukemia (ATL) HTLV-I-associated myelopathy⁄tropical spastic paraparesis (HAM/TSP). Golestan province is located in North West of Khorasan province known as an endemic area for HTLV-I in Iran. This study aimed to evaluate seroprevalence of HTLV-I in Golestan province.Materials and Methods: In this cross-sectional descriptive study in 2007, blood samples were collected from 2034 healthy people residing in different parts of Golestan province. Sera were assessed for HTLV-I/II–specific antibodies by ELISA method and reactive samples were confirmed by Western blot. Demographic and serologic data were entered in SPSS version 11.5 and statistical analysis was performed.Results: An overall HTLV-I/II prevalence of 0.7% was observed in 15 cases by ELISA. Six out of 15 were confirmed as HTLV-I by western blot. Regional variation in the prevalence of HTLV-I was observed; 0%, 0%, 0.1%, 1.9%, 0.3%, 0%, and 2.6% tested HTLV-I-positive from west to east of Golestan Province regions, respectively. Seropositivity increased with age. No association between HTLV-I infection and sex status was detected.Conclusion: Highest rate of HTLV-I seroprevalence was shown in east of this region located in neighborhood with Khorasan province, the only confirmed endemic area in Iran. It seems that eastern area of our province is endemic for HTLV-I. Further comprehensive detailed epidemiological and molecular studies are recommended. Human T-cell lymphotropic virus type-1 (HTLV-I) is a member of Retroviridae family which has been discovered as the first human retrovirus. This oncornavirus causes the T cell malignancy associated with two main diseases; adult T cell leukemia (ATL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) . WorldwiIran has been introduced as an endemic area based on studies reported from Mashhad in Khorasan (A province of Iran recently divided into three provinces) located in the northeast of Iran , 12. The◦C. Serum samples were screened for the presence of anti-HTLV-I antibodies with the HTLV I/II enzyme linked immunosorbent assay (ELISA)  according to the manufacturer’s instructions. All reactive samples on serologic screening were tested further through Western blot (WB) analysis according to the manufacturer’s instructions . Descriptive data was summarized as the mean, standard deviation, and/or percentages were analyzed by SPSS 11.5 using Chi-square and t-tests. A P-Value of <0.05 was considered statistically significant.From all of the seven main cities with an estimated population of 1.5 million, 2034 individuals were selected through multistage cluster sampling in 2007. Demographic information, such as sex, age, and residency status, was collected. The study was approved by Deputy of Research of Golestan University of Medical Sciences regarding scientific and ethical issues. Informed consent was obtained from all participants. Five ml of blood samples were obtained from each individual. Serum was separated through centrifugation and was stored at −20Out of 2034 people studied in this population, based seroepidemiology, 848 cases were male (41.7%) and 1186 participants were females (58.3%) with the mean age of 38.66±16.54 years. All serum samples were analyzed for anti-HTLV antibodies.In the primary screening of the samples by ELISA, 15 (0.7%) were positive for HTLV1/2 antibodies. The Western blot results confirmation demonstrated that 6 (0.3%) out of 15 ELISA positive specimens were HTLV-1 positive, but HTLV positivity was not confirmed in 9 cases (0.4%). According to the Western blot results, the overall prevalence of the HTLV-I infection in the population under study was 0.3% (6/2034). We did not find an indeterminate result by Western blot. The HTLV-I infection rate for females was 0.3% (3/1186) and for males was 0.4% (3/848). No significant difference in the seroprevalence was observed between males and females .Regional variation in the prevalence of HTLV-I was observed to be 0%, 0% 0.1%, 1.9%, 0.3%, and 2.6% tested HTLV-I-positive from west to east of Golestan regions, respectively . SeropreIn the present study, we have reported the seroepidemiology of HTLV-I infection in a representative sample of individuals from different cities of the province of Golestan. The overall prevalence of HTLV-I infection in our study is 0.3% (95% CI: 0.06-0.53%) lower than the previous study with 4.4% in Thalassemia patients in Gorgan . This isfrom different investigations in the USA 0.6% , Brazil Comparison of the regional distribution of the carriers in the present study revealed a significant increase of the HTLV-I carriers in Kalaleh area. The observed changes could be considered mainly due to the migration of people from other areas. This interpretation is supported by the observation of ethno-epidemiology studies on HTLV-I carriers who are either born in the endemic areas or the descendants of migrants from those areas. It has been reported that some ethnically defined factors are likely to be associated with HTLV-I diseases among HTLV-I endemic populations .Previous studies have demonstrated that HTLV-I infection increases with age -29. AlsoIn our study, the age distribution of carriers showed presence of HTLV-I carrier in age groups more than 20 years old and the largest number of carriers was observed in this age group. Recent studies showed the number of carriers in the age groups between 0–9 and 50–59 significantly decreased . This deIn conclusion, serological screening of pregnant women, and the blood donors, along with the prevention of mother-to-child infection transmission by stopping breast feeding will greatly reduce the vertical transmission. In addition, there still remain other modalities of HTLV-I infection, such as sexual transmission and possible transuterine infection -37. Furt


All animals received both contrast agents on separate days. Selective intra-arterial x-ray angiograms (XRAs) were obtained in the ligated limb as a reference. The number of collaterals was counted by two independent observers. Image quality was evaluated with the contrast-to-noise ratio (CNR) in the femoral artery and collateral arteries. CNR for Gadomer was higher in both the femoral artery (Gadomer: 73±5 (mean ± SE); Gd-DTPA: 40±3; p<0.01) and collateral arteries . Neither day of acquisition nor contrast agent used influenced the number of identified collateral arteries . An average of 4.5±1.0  and 5.3±1.2 (day 21) collaterals was found, which was comparable to XRA . Inter-observer variation was 24% and 18% for Gadomer and Gd-DTPA, respectively. In conclusion, blood pool agent Gadomer improved vessel conspicuity compared to Gd-DTPA. Steady-state MRA can be considered as an excellent non-invasive alternative to intra-arterial XRA for the visualization of ultra-small collateral arteries.The objective of this study was to compare the blood pool agent Gadomer with a small contrast agent for the visualization of ultra-small, collateral arteries (diameter<1 mm) with high resolution steady-state MR angiography (SS-MRA) in a rabbit hind limb ischemia model. Ten rabbits underwent unilateral femoral artery ligation. On days 14 and 21, high resolution SS-MRA (voxel size 0.49×0.49×0.50 mm Therapeutic stimulation of the development of collateral arteries from pre-existent arterioles (arteriogenesis) seems attractive as an alternative or adjuvant treatment for patients with peripheral arterial occlusive disease Therapeutic efficacy can be evaluated functionally, i.e. by measuring perfusion recovery-related parameters in tissue distal to the vascular lesion, or morphologically, i.e. by quantifying the number and size of the collaterals formed. Contrast-enhanced MR angiography (CE-MRA) has already proven to be a promising non-invasive tool to visualize collateral arteries in various parts of the body T1 relaxivity compared to small contrast agents because of their size An alternative is to acquire data during the post-bolus equilibrium phase or steady state, which permits longer acquisition times and therefore a higher spatial resolution. Due to its longer acquisition time, steady-state MRA (SS-MRA) is less sensitive for timing uncertainties than first-pass MRA (FP-MRA), as demonstrated for the visualization of arterial stenoses in patients with peripheral arterial occlusive disease Various types of types of gadolinium-based BPAs have been developed. One group consists of small BPAs that non-covalently bind to plasma proteins, with the albumin-binding gadofosveset trisodium as its principal, clinically approved example In this study, we compared the blood pool agent Gadomer to the small contrast agent Gd-DTPA for the visualization of small collateral arteries with steady-state contrast-enhanced MRA in a standardized rabbit hind limb ischemia model exhibiting arteriogenesis. For both contrast agents, image quality characteristics and number of visible collaterals were determined and compared to intra-arterial XRA.This study was approved by the Maastricht University animal ethics committee . Ten male New Zealand White rabbits (weight: approximately 3 kg) were included. Occlusion of the right femoral artery was realized by placing ligations 1 cm below the branch of the circumflex femoral artery (CFA) and 1 cm above the bifurcation of the popliteal and tibial arteries.For all procedures, anesthesia was induced by intramuscular injection of a combination of ketamine hydrochloride (50 mg/kg body weight) and xylazin (8 mg/kg body weight). During the procedures, the rabbits were intubated and ventilated with isoflurane (2–3%) and oxygen. Post-operative analgesia was provided during three days by daily intramuscular injections of buprenorphine (0.09 mg/kg body weight). All invasive procedures were performed under sterile conditions. After the last MR measurements a lethal dose of pentobarbital (1 mL/kg body weight) was administered intravenously.−1. An intra-arterial catheter was inserted into the left carotid artery and positioned in the iliac artery of the ligated limb, where a 5 mL bolus of non-ionic iodine contrast agent  was injected at 5 mL/s, followed by a saline flush.On days 14 and 21 post ligation, XRA series of anterior-posterior projections were recorded using a portable x-ray system . In-plane resolution was 0.2×0.2 mm, tube voltage was 72 kV and frame rate was 12 sWithin one hour after XRA, the animals were imaged in supine position on a clinical 3.0 Tesla MRI system equipped with a 5-element phased-array cardiac coil . The rabbits were placed in a custom-made fixating box to standardize animal positioning.T1-weighted spoiled fast gradient echo sequence. Field-of-view (FOV) in the cranio-caudal read-out direction was 250 mm and rectangular FOV was 80%. Parallel imaging acceleration (SENSE) factor in the right-left direction was 2. Acquisition was started after a delay of two minutes. Matrix size was 512×410 with 282 overlapping coronal slices (slice thickness 0.5 mm). Measured voxel size was 0.49×0.49×0.50 mm3. TR/TE/FA were 10.5 ms/2.7 ms/25°. Acquisition time was 5 minutes.The steady-state MRA (SS-MRA) protocol consisted of a 3+ ions. It has a molecular weight of 17 kDa. Gd-DTPA and Gadomer were administered in a randomized order on separate days. The contrast agent  was injected at 0.05 mL/s into an ear vein, followed by 2 mL saline flush injected at the same rate.To all animals both a small extravascular agent (Gd-DTPA) and a medium-sized blood pool agent (Gadomer) were administered. Gadomer  is a dendritic gadolinium chelate, containing 24 GdThe contrast agent dose was selected by estimating signal enhancement during the course of SS-MRA acquisition based on a biexponential approximation of contrast agent concentration determined in a previous study From the dynamic XRA image series a maximum intensity projection (MIP) was calculated in MATLAB  over the time frames that exhibited arterial enhancement. The resulting images were used for collateral identification. The MR angiograms were viewed in the image processing application OsiriX (version 3.7), using the source images for signal intensity measurements. For collateral identification, coronal maximum intensity projections over a limited range of slices  were used. The thickness and location of the slab, as well as the display contrast and brightness levels could be adjusted in real time to obtain optimal depiction of the collateral trajectories and varied depending on image quality and vascular anatomy.The number of collaterals was counted on the partial MIP by two independent observers using the Longland definition Arteries were separated from veins based on their size, location, and branching point. Due to evident differences in image quality, the observers were not blinded for contrast agent.muscle). From these data signal-to-noise (SNR) and contrast-to-noise (CNR) ratios were calculated according to:Signal intensities (SI) were measured in the femoral artery , a collateral artery stemming from the DFA, and in muscle tissue surrounding the collateral. Noise was estimated by calculating the standard deviation of a relatively homogeneous part of adjacent muscle tissue  was used to test whether day of acquisition or contrast agent used (for MRA) had an influence on the number of identified collateral arteries. Independent sample two-sided Student's On the steady-state MR angiograms, both arteries and veins were enhanced, but the spatial configuration of the vessels and the high spatial resolution of the angiograms allowed distinction of arteries from veins see  and S2. p<0.01), and had the same, yet statistically non-significant trend in the collateral arteries (p = 0.10). CNR was significantly higher in both femoral and collateral arteries when Gadomer was used . ROI size was typically 10 voxels in the femoral artery and 3 voxels in the collateral artery. Noise and background signal was measured over an area of approximately 3 cm2.SNR was higher in both the femoral and collateral arteries for Gadomer compared to Gd-DTPA . This imp = 0.30 and p = 0.14, respectively). Although the animals were positioned in a custom-made fixating box, it was not feasible to perform 1-to-1 comparison of the collaterals found with either contrast agent due to difficulties in determining the exact course of the collateral arteries. Inter-observer variation in the number of identified collaterals on SS-MRA was 24% and 18% for Gadomer and Gd-DTPA, respectively, meaning that the difference between observers is approximately one collateral artery. Identification of the exact points of branching and re-entering appeared to be highly subjective, and no conclusions could be drawn from the schematic vessels courses.p = 0.04). The number of collateral arteries found with XRA was equal to the number found with MRA (p = 0.08). For XRA, most collaterals originated from the deep femoral arteries (120 out of 172 or 70%), whilst for MRA only 50% (77 out of 153) stemmed from the DFA.The number of collateral arteries found with XRA increased between days 14 and 21 (day 14: 5.1±1.7 (mean ± SD); day 21: 6.3±1.8; To our knowledge, this is the first study that directly compares the efficacy of the blood pool agent Gadomer with Gd-DTPA for the visualization of ultra-small collateral arteries. The number of collaterals identified on the steady-state images was equal for the two contrast agents, and closely mirrored the number of collaterals identified on high resolution invasive X-ray angiograms. Contrast enhancement in collateral arteries in terms of CNR and therefore vessel conspicuity, was superior for Gadomer compared to Gd-DTPA.The use of Gadomer resulted in a considerably higher contrast between collateral arteries and background compared to Gd-DTPA in the SS-MRA images. Because of this improved image quality, thick-slab MIPs could be used to get a better overview of the vessel trajectories, which notably facilitated the localization of collateral arteries, thus making it less time-consuming. For Gd-DTPA, the value of these thick-slab MIPs was limited, because background enhancement became problematic for slabs thicker than approximately 8 slices (5 mm). Moreover, thick-slab MIPs may suggest connections between vessels which are in fact crossings of different vessels. Therefore, to follow the trajectory of the collateral artery from stem to re-entry zone, thin-slab MIPs were used, which combined the favorable contrast-to-noise characteristics of source images with the improved overview related to MIPs. Using thin-slab MIPs for the actual identification diminished the disadvantage for Gd-DTPA, as the contrast between collaterals and background was still high enough to identify an equal number of collaterals compared to XRA, leaving little room for improvement with Gadomer.r1 value for Gadomer.One of the main advantages of blood pool agents is their decreased extravasation. For Gadomer, the extravasation rate is approximately 10 times lower compared to Gd-DTPA r1  and a lower r2 , which may favor Gadomer for imaging at 3 Tesla.Although Gadomer has already been safely used for angiography in humans In addition to the advantages regarding invasiveness and ionizing radiation, MRA has the advantage of the availability of 3D data compared to 2D XRA, which is a one-directional projection. On these projections no distinction can be made between arteries that branch off or cross other arteries, which hinders the identification of stem and re-entry zones. A second disadvantage of XRA is the high signal from bone structures. In clinical practice, this problem is usually solved by using digital subtraction of pre- and post-contrast images. In our study, vasospasms and animal movement due to contrast injection regularly resulted in blurred images which were less suited for collateral identification. Consequently, the proximity of the femur to the mid and re-entry zone of collaterals stemming from the circumflex femoral artery resulted in the identification of fewer collaterals stemming from the CFA on XRA images compared to MRA. Although the spatial resolution achieved with MRA is generally lower than with XRA, MRA compared well to XRA for both contrast agents and the total number of collaterals visualized on MRA was comparable to that of XRA.The blood pool agent Gadomer has great potential in the visualization of small collateral arteries with steady-state contrast-enhanced MRA. The total number of identified collaterals was equal to XRA. Although no difference between Gadomer and Gd-DTPA in number of identified collaterals or interobserver variability was found in this study, the enhanced collateral artery conspicuity appreciably facilitated the identification of collateral arteries with Gadomer compared to Gd-DTPA, while the dose was reduced.Figure S1Signal enhancement time course. Signal enhancement time course for a range of contrast agent doses for Gadomer  and Gd-DTPA . Grey shaded regions indicate the acquisition window for steady-state MRA in this study.(TIF)Click here for additional data file.Appendix S1Dose optimization. Procedure for the selection of the optimal dose for Gadomer and Gd-DTPA.(DOC)Click here for additional data file.Video S1MRA with Gadomer. Dynamic overview of a typical MRA data set with Gadomer. Partial maximum intensity projections in the anterior-posterior direction of steady-state MR angiograms (SS-MRA) were obtained at varying positions. Slab thickness was 2 cm.(AVI)Click here for additional data file.Video S2MRA with Gd-DTPA. Dynamic overview of a typical MRA data set with Gd-DTPA. Partial maximum intensity projections in the anterior-posterior direction of steady-state MR angiograms (SS-MRA) were obtained at varying positions. Slab thickness was 2 cm.(AVI)Click here for additional data file.